Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art which are analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Pat. Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, and 5,174,993, the disclosures of which are incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome. Additional strategies have recently been reported for generating recombinant vaccinia virus.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. In the course of its history, many strains of vaccinia have arisen. These different strains demonstrate varying immunogenicity and are implicated to varying degrees with potential complications, the most serious of which are post-vaccinial encephalitis and generalized vaccinia (Behbehani, 1983).
With the eradication of smallpox, a new role for vaccinia became important, that of a genetically engineered vector for the expression of foreign genes. Genes encoding a vast number of heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990a, 1990b).
The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain of vaccinia virus was protective (Morgan et al., 1988).
A fine balance between the efficacy and the safety of a vaccinia virus-based recombinant vaccine candidate is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated animal but lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology.
A number of vaccinia genes have been identified which are non-essential for growth of the virus in tissue culture and whose deletion or inactivation reduces virulence in a variety of animal systems.
The gene encoding the vaccinia virus thymidine kinase (TK) has been mapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir et al., 1983). Inactivation or complete deletion of the thymidine kinase gene does not prevent growth of vaccinia virus in a wide variety of cells in tissue culture. TK.sup.- vaccinia virus is also capable of replication in vivo at the site of inoculation in a variety of hosts and administered by a variety of routes.
It has been shown for herpes simplex virus type 2 that intravaginal inoculation of guinea pigs with TK.sup.- virus resulted in significantly lower virus titers in the spinal cord than did inoculation with TK.sup.+ virus (Stanberry et al., 1985). It has been demonstrated that herpesvirus encoded TK activity in vitro was not important for virus growth in actively metabolizing cells, but was required for virus growth in quiescent cells (Jamieson et al., 1974).
Attenuation of TK.sup.- vaccinia has been shown in mice inoculated by the intracerebral and intraperitoneal routes (Buller et al., 1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK.sup.- recombinant vaccinia generated equivalent anti-vaccinia neutralizing antibodies as compared with the parental TK.sup.+ vaccinia virus, indicating that in this test system the loss of TK function does not significantly decrease immunogenicity of the vaccinia virus vector. Following intranasal inoculation of mice with TK.sup.- and TK.sup.+ recombinant vaccinia virus (WR strain), significantly less dissemination of virus to other locations, including the brain, has been found (Taylor et al., 1991a).
Another enzyme involved with nucleotide metabolism is ribonucleotide reductase. Loss of virally encoded ribonucleotide reductase activity in herpes simplex virus (HSV) by deletion of the gene encoding the large subunit was shown to have no effect on viral growth and DNA synthesis in dividing cells in vitro, but severely compromised the ability of the virus to grow on serum starved cells (Goldstein et al., 1988). Using a mouse model for acute HSV infection of the eye and reactivatable latent infection in the trigeminal ganglia, reduced virulence was demonstrated for HSV deleted of the large subunit of ribonucleotide reductase, compared to the virulence exhibited by wild type HSV (Jacobson et al., 1989).
Both the small (Slabaugh et al., 1988) and large (Schmidtt et al., 1988) subunits of ribonucleotide reductase have been identified in vaccinia virus. Insertional inactivation of the large subunit of ribonucleotide reductase in the WR strain of vaccinia virus leads to attenuation of the virus as measured by intracranial inoculation of mice (Child et al., 1990).
The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced (Shida, 1986). The HA gene of vaccinia virus is nonessential for growth in tissue culture (Ichihashi et al., 1971). Inactivation of the HA gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA.sup.- vaccinia virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987).
Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks on the chorioallantoic membrane of chicken eggs. Spontaneous deletions within the cowpox genome generate mutants which produce white pocks (Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDa protein encoded by an early gene (Pickup et al., 1986). This gene, which has homology to serine protease inhibitors, has been shown to inhibit the host inflammatory response to cowpox virus (Palumbo et al., 1989) and is an inhibitor of blood coagulation.
The u gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Mice inoculated with a WR vaccinia virus recombinant in which the u region has been inactivated by insertion of a foreign gene produce higher antibody levels to the foreign gene product compared to mice inoculated with a similar recombinant vaccinia virus in which the u gene is intact (Zhou et al., 1990). The u region is present in a defective nonfunctional form in Copenhagen strain of vaccinia virus (open reading frames B13 and B14 by the terminology reported in Goebel et al., 1990a,b).
Cowpox virus is localized in infected cells in cytoplasmic A type inclusion bodies (ATI) (Kato et al., 1959). The function of ATI is thought to be the protection of cowpox virus virions during dissemination from animal to animal (Bergoin et al., 1971). The ATI region of the cowpox genome encodes a 160 kDa protein which forms the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987). Vaccinia virus, though containing a homologous region in its genome, generally does not produce ATI. In WR strain of vaccinia, the ATI region of the genome is translated as a 94 kDa protein (Patel et al., 1988). In Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI region are deleted, with the remaining 3' end of the region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 1990a,b).
A variety of spontaneous (Altenburger et al., 1989; Drillien et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicali et al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989; Perkus et al., 1986) deletions have been reported near the left end of the vaccinia virus genome. A WR strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al., 1988b). Specific genes within the deleted region include the virokine N1L and a 35 kDa protein (C3L, by the terminology reported in Goebel et al., 1990a,b). Insertional inactivation of N1L reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly the complement 4B binding protein (C4bp) (Kotwal et al., 1988a). Like the cellular C4bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms.
The left end of the vaccinia genome includes two genes which have been identified as host range genes, K1L (Gillard et al., 1986) and C7L (Perkus et al., 1990). Deletion of both of these genes reduces the ability of vaccinia virus to grow on a variety of human cell lines (Perkus et al., 1990).
Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipox viruses. Both fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.
FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990).
Despite the host-restriction for replication of FPV and CPV to avian systems, recombinants derived from these viruses were found to express extrinsic proteins in cells of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993a,b; Taylor et al., 1992; 1991b; 1988b).
Feline infectious peritonitis virus (FIPV) produces a chronic, progressive, immunologically-mediated disease in felines such as domestic and exotic cats. The route of FIPV infection is thought to occur primarily through the oral cavity and pharynx. Clinically apparent FIP occurs after the virus crosses the mucosal barrier and a primary viremia takes FIPV to its many target organs (liver, spleen, intestine and lungs). Two forms of the disease have been described as effusive (wet) and non-effusive (dry). The effusive form results in the classic fluid accumulation seen in infected cats which is caused by an Arthus-type vasculitis in the target organs mediated by complement activation and an intense inflammatory response. The non-effusive form is characterized by little or no ascitic fluid accumulation but internal organs may be infiltrated with granular fibrinous deposits. Thus, antibodies formed in response to FIPV infection (primarily to the spike protein) tend to enhance the pathogenesis of the disease and are obviously unwanted in a vaccine or immunological composition (Olsen and Scott, 1991). (However, expression of such proteins by a recombinant and the recombinants themselves are useful if one desires antigens or antibodies therefrom for a kit, test or assay or the like).
FIPV is a member of the Coronaviridae family. Coronaviruses are large, positive stranded RNA viruses with genomic lengths of 27-30 kb. The virion is enveloped and is studded with peplomeric structures called spikes. The left half of the FIPV genome encodes a large polyprotein which is cleaved into smaller fragments, some of which are involved in RNA replication. The right half of the FIPV genome encodes 3 major structural proteins designated nucleocapsid (N), matrix (M) and spike (S). The FIPV S gene product mediates attachment of the virus to the cell receptor, triggers membrane fusion, and elicits virus-neutralizing antibodies. The N protein is necessary for encapsidating genomic RNA and directing its incorporation into the capsid, and is thought to be involved in RNA replication. The FIPV M glycoprotein appears to be important for FIP viral maturation and for the determination of the site at which virus particles are assembled (Spann et al., 1988).
Because of the antibody-dependent enhancement (ADE) of FIP in cats, attempts to produce a safe and efficacious vaccine or immunological composition against FIPV have been largely unsuccessful. Inactivated FIPV vaccines and heterologous live coronavirus vaccines did not afford any protection against FIPV infection and vaccination usually resulted in increased sensitization to the disease. A modified live virus vaccine, Primucell, is the first and only commercially marketed FIPV vaccine. Primucell is a temperature sensitive strain of FIPV that can replicate at the cooler temperatures of the nasal cavity, but not at systemic body temperatures (Gerber et al., 1990). Thus, intranasally administered Primucell is thought to produce a localized immunity to FIPV. However, serious questions remain concerning the efficacy and enhancement potential of this vaccine (Olsen and Scott, 1991).
Vaccinia virus has been used as a vector for generating recombinant viruses expressing FIPV structural genes. A recombinant expressing the FIP M gene was shown to increase the survival time of cats after challenge with FIPV (Vennema et al., 1990).
Vennema, et al. (1991) relates to primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and to certain recombinant vaccinia viruses thereof introduced into kittens. The Vennema et al. FIPV matrix gene was cloned from a pathogenic strain (79-1146) and its sequence appears identical to the matrix gene (discussed herein). The Vennema et al. recombinant, vFM, contains the coding region of matrix coupled to the vaccinia 7.5K early/late promoter inserted at the thymidine kinase (tk) locus. Note that the promotor was not linked precisely to the matrix ATG initiation codon, but rather to a position 48 bp upstream from the ATC. Also, a vaccinia T5NT early transcriptional termination signal (Yuen et al., 1987) located in the coding region of the matrix gene was not removed.
Moreover, the vaccinia strain in Vennema et al. is the WR strain (Vennema et al. at page 328, left column, first 2 lines; see also, the donor plasmids and control viruses as mentioned on the same page in the section "Construction of Recombinant Vaccinia Viruses expressing the FIPV M and N proteins" beginning at mid-left column clearly indicate via literature citations that the WR strain is used). The choice of strain is important because the WR strain is a laboratory virus--not a vaccine strain--and the virulence characteristics of the WR strain do not make it a presently acceptable vector for a recombinant that may contact humans, let alone a recombinant in a composition such as a vaccine or antigenic or immunological composition targeted to felines, such as kittens, or other animals in contact with humans, especially young children or immunosuppressed individuals, due to recent concerns of contact transmission (such "other animals" could be laboratory cell cultures or animals for antigen expression or for antibody production for making kits, tests or assays).
Thus, the Vennema, et al. articles fail to teach or suggest the recombinants, compositions and methods of the present invention.
More particularly, recombinants in the present invention preferably employ NYVAC or vectors (NYVAC and ALVAC are highly attenuated vectors having a BSL1 containment level).
Further, in constructs of the present invention, preferably the coding region is coupled to the promotor in a precise coupling to the ATG codon with no intervening sequence. (Any T5NT sequence can be inactivated by a base substitution which does not change the amino acid sequence but will prevent early transcriptional termination in a poxvirus vector). In addition, multiple, e.g., two, copies of the coding region directly coupled to the promotor can be present in each recombinant viral genome in the present invention.
The Vennema et al. efficacy study used SPF kittens (13-14 weeks old) which were vaccinated subcutaneously at day 0 and day 21 with 1.times.10.sup.8 and 5.times.10.sup.8 pfu respectively. On day 35 the cats were challenged orally with FIP strain 79-1146.
The herein protocol was similar, with the major difference being a lower vaccination dose (1.times.10.sup.7). The Vennema protection results were based on mortality with 3 of 8 cats vaccinated with vFM surviving (37.5%). Vennema et al. deemed their challenge sufficient in that 7 of 8 unvaccinated cats succumbed to the challenge exposure and died. Upon necropsy, all challenged cats, in Vennema et al. including the three surviving vFM vaccinated cats, had pathological signs of FIP infection including peritoneal effusions and granulomatous lesions on the viscera.
By contrast, the trials herein were more stringent. Herein applicants scored protection as surviving and being free from FIP pathology upon necropsy. Using this criteria, Applicants had 3 out of 5 cats vaccinated with vCP262 protected (60%) with 0% of the unvaccinated cats protected.
If the Vennema et al. results were scored using Applicants' criteria, Vennema would have had no protection; and ergo no recombinant suitable for vaccine use. In addition, the Vennema et al. observed fever and weight loss in all challenged cats. In Applicants' trials, (see trial 3 in particular) Applicants' observed even no weight loss and a lower febrile response after challenge.
Thus, the recombinants of the present invention employ an acceptable vector for all uses and a surprisingly higher protection level at a lower dose than the Vennema et al. vaccinia recombinant.
Recent studies using monoclonal antibodies directed against the S gene (Olsen et al., 1992) have shown also that mABs which neutralize the virus also cause ADE. No enhancement is observed with mABs against matix or nucleocapsid proteins.
Thus, prior to the present invention, there has been a need for poxvirus-FIPV recombinants, especially such recombinants using an acceptable vector and such recombinants having expression at low doses which indeed affords protection; and, there has been a need for compositions containing such recombinants, as well as a need for methods for making and using them. And, moreover, it would be especially surprising and unexpected if this poxvirus-FIPV recombinant was modified so as to be attenuated, e.g., an attenuated vaccinia virus-FIPV recombinant or an attenuated avipox-FIPV recombinant, such as a NYVAC-FIPV or ALVAC-FIPV recombinant; because, for instance, from attenuation and, diminished or lack of productive replication of the poxvirus in the host, one skilled in the art would have not expected and would be surprised by the usefulness of the attenuated recombinant, especially in a composition for felines and other hosts, and more especially in such a composition which provides a response including protection in felines.
Attenuated poxvirus vectors would also be especially advantageous for antigenic or vaccine compositions, particularly in view of attenuated vectors providing diminished or little or no pathogenic properties with regard to the intended host or, to unintended, possibly accidental hosts, such as those who work with the vector in formulating or administering the vector or antigen, or who may otherwise come into contact with it. That is, attenuated poxvirus vectors provide diminished or little or no pathogenic properties to intended hosts such as cats, kittens and the like and to unintended, possibly accidental hosts, such as humans engaged in formulating the vector into a composition for administration or in administering the composition (e.g., veterinarians, technicians, other workers) or, who may otherwise come into contact with the vector (e.g., pet owners).
It can thus be appreciated that provision of a FIPV recombinant poxvirus, and of compositions and products therefrom, particularly NYVAC or ALVAC based FIPV recombinants and compositions and products therefrom, would be a highly desirable advance over the current state of technology.